Streptococci species possess a multitude of adhesins on their cell surfaces that facilitate adherence to a wide range of substrates; this is the first sep necessary for the development of disease. The most far-reaching example of disease is dental caries and inflammatory periodontal disease since the main portal of entry into the human body is through the oral cavity. A large portion of bacterial species in the oral cavity are the sanguis streptococci (S. sanguis, S. gordonii, S. oralis, S. parasanguinis, and related species). As one of the first colonizers of the tooth surface, not only do sanguis streptococci interact and adhere to host epithelial cells, but they also act as a necessary platform for other microbial cells to attac and form a biofilm, including those that cause periodontal diseases. Therefore, the initial binding of sanguis streptococci to the oral cavity has significant implications for the development of periodontal diseases. S. parasanguinis adhesion to an in vitro tooth surface model, saliva-coated hydroxyapatite, is mediated by long peritrichous fimbriae. These fimbriae contain a 200 kDa surface glycoprotein, Fap1, which is involved in bacterial adhesion, colonization, and biofilm formation. Thus, the investigation of Fap1 is important to understanding the dynamics of bacterial attachment. While the non-glycosylated Fap1 peptide mediates initial attachment during the process of biofilm formation, glycosylation of Fap1 (O-linkage to a multitude of serine residues) is involved in fimbrial assembly, bacterial adhesion, biofilm formation, and adherence to the oral cavity. An eleven gene cluster encoding glycosyltransferases and accessory Sec components is required for Fap1 biogenesis and glycosylation and includes a core region that is conserved in every genome (secY2, gap1-3, secA2, and gtf1-2) and a variable region (gly, nss, galT1, and galT2). The fap1 biogenesis cluster is very conserved among streptococci and staphylococci; since Fap1-like proteins are implicated in bacterial pathogenesis, our studies may reveal new targets amenable for drug discovery. The exact mechanism of how Fap1 is glycosylated remains a mystery. We have been particularly interested in the three glycosylation-associated proteins- Gap1, Gap2, and Gap3. Knock-outs of all three genes produce a Fap1 protein that is incompletely glycosylated or improperly folded, suggesting that these three genes are involved in Fap1 biogenesis. The interaction between Gap1 and Gap3 has been shown to be required for Fap1 biogenesis. To date, the function of Gap2 is still unknown. I hypothesize that Gap2 is involved in the biogenesis of Fap1 through its interactions with Gap1, Gap3, and other gene products necessary for Fap1 biogenesis. To test this hypothesis, I propose to investigate these specific aims: 1) assess the biochemical function of Gap2; 2) analyze Gap2 interaction with other proteins in the Fap1 cluster; 3) determine the role Gap2 plays in Fap1 biogenesis. PUBLIC HEALTH RELEVANCE: The most far-reaching example of streptococcal disease is dental caries and inflammatory periodontal disease since the main portal of entry into the human body is through the oral cavity. As one of the first colonizers of the tooth surface and a large portion of the bacterial species in the oral cavity, sanguis streptococci not only interact ad adhere to host epithelial cells, but also act as a necessary platform for other microbial cells (including those involved in periodontal diseases) to attach and form a biofilm. As a sanguis streptococci, Streptococcus parasanguinis plays an important role in the formation of dental plaque.